Genetic regulation of plasma von Willebrand factor levels in health and disease.

Plasma levels of the multimeric glycoprotein von Willebrand factor (VWF) constitute a complex quantitative trait with a continuous distribution and wide range in the normal population (50-200%). Quantitative deficiencies of VWF (< 50%) are associated with an increased risk of bleeding, whereas high plasma levels of VWF (> 150%) influence the risk of arterial and venous thromboembolism. Although environmental factors can strongly influence plasma VWF levels, it is estimated that approximately 65% of this variability is heritable. Interestingly, although variability in VWF can account for ~ 5% of the genetic influence on plasma VWF levels, other genetic loci also strongly modify plasma VWF levels. The identification of the additional sources of VWF heritability has been the focus of recent observational trait-mapping studies, including genome-wide association studies or linkage analyses, as well as hypothesis-driven research studies. Quantitative trait loci influencing VWF glycosylation, secretion and clearance have been associated with plasma VWF antigen levels in normal individuals, and may contribute to quantitative VWF abnormalities in patients with a thrombotic tendency or type 1 von Willebrand disease (VWD). The identification of genetic modifiers of plasma VWF levels may allow for better molecular diagnosis of type 1 VWD, and enable the identification of individuals at increased risk for thrombosis. Validation of trait-mapping studies with in vitro and in vivo methodologies has led to novel insights into the life cycle of VWF and the pathogenesis of quantitative VWF abnormalities.

Early cellular interactions and immune transcriptome profiles in human factor VIII-exposed hemophilia A mice.

Essentials Initial immune cell interactions leading to factor (F) VIII immunity are not well characterized. We assessed cellular interactions and expression profiles in hemophilia A mice. MARCO+, followed by SIGLEC1+ and SIGNR1+ macrophages co-localize most with human FVIII. The splenic transcriptome highlights potential therapeutic targets to prevent inhibitors. SUMMARY: Background Developing factor VIII (FVIII) inhibitory antibodies is the most serious complication in hemophilia A treatment, representing a significant health and economic burden. A better understanding of the early events in an immune response leading to this outcome may provide insight into inhibitor development. Objective To identify early mediators of FVIII immunity and to detail immune expression profiles in the spleen and liver. Methods C57Bl/6 F8 E16 knockout mice were infused with 5-20 µg (2000-8000 IU kg-1 ) of recombinant FVIII. Spleens were frozen at various time-points post-infusion and stained for FVIII and cellular markers. Splenic and liver RNA expression analysis was performed 3 h post-infusion of 0.6 µg (240 IU kg-1 ) FVIII by nCounter technology using a 561-gene immunology panel. Results FVIII localization in the spleen did not change over 2.5 h. We observed significantly higher co-localization of FVIII with MARCO+ cells compared with SIGLEC1+ and SIGNR1+ in the splenic marginal zone. FVIII exhibited little co-localization with CD11c+ dendritic cells and the macrophage mannose receptor, CD206. Following FVIII infusion, the splenic mRNA profiling identified genes such as Tnfaip6 and Il23r, which are implicated in chemotaxis and a proinflammatory Th17 response, respectively. In contrast, an upregulation of Gfi1 in the liver suggests an anti-inflammatory environment. Conclusions FVIII co-localizes predominantly with marginal zone macrophages (MARCO+ ) in the murine spleen following intravenous infusion. Targeting pathways that are implicated in the early FVIII innate immune response in the spleen may lead to therapeutic interventions to prevent inhibitor formation.

Abnormal von Willebrand factor secretion, factor VIII stabilization and thrombus dynamics in type 2N von Willebrand disease mice.

Essentials Type 2N von Willebrand disease involves impaired von Willebrand factor to factor VIII binding. Type 2N von Willebrand disease mutations exhibit qualitative and mild quantitative deficiencies. Type 2N von Willebrand disease mice exhibit unstable venous hemostatic thrombi. The factor VIII-binding ability of von Willebrand factor regulates arteriole thrombosis dynamics. SUMMARY: Background von Willebrand factor (VWF) and factor VIII (FVIII) circulate as a non-covalent complex, with VWF serving as the carrier for FVIII. VWF indirectly influences secondary hemostasis by stabilizing FVIII and transporting it to the site of primary hemostasis. Type 2N von Willebrand disease involves impaired binding of VWF to FVIII, resulting in decreased plasma levels of FVIII. Objectives In these studies, we characterize the impact of three type 2N VWD variants (R763A, R854Q, R816W) on VWF secretion, FVIII stabilization and thrombus formation in a murine model. Methods Type 2N VWD mice were generated by hydrodynamic injections of mutant murine VWF cDNAs and the influence of these variants on VWF secretion and FVIII binding was evaluated. In vivo hemostasis and the dynamics of thrombus formation and embolization were assessed using a murine tail vein transection hemostasis model and an intravital thrombosis model in the cremaster arterioles. Results Type 2N VWD variants were associated with decreased VWF secretion using cell and animal-based models. FVIII-binding to type 2N variants was impaired in vitro and was variably stabilized in vivo by expressed or infused 2N variant VWF protein. Both transgenic type 2N VWD and FVIII knockout (KO) mice demonstrated impaired thrombus formation associated with decreased thrombus stability. Conclusions The type 2N VWD phenotype can be recapitulated in a murine model and is associated with both quantitative and qualitative VWF deficiencies and impaired thrombus formation. Patients with type 2N VWD may have normal primary hemostasis formation but decreased thrombus stability related to ineffective secondary hemostasis.

Histones link inflammation and thrombosis through the induction of Weibel-Palade body exocytosis.

Essentials Dysregulated DNA and histone release can promote pathological immunothrombosis. Weibel-Palade bodies (WPBs) are sentinel-like organelles that respond to proinflammatory stimuli. Histones induce WPB exocytosis in a caspase, calcium and charge-dependent mechanism. A targetable axis may exist between DNA/histones and WPBs in inflammation and immunothrombosis. SUMMARY: Background Damage-associated molecular patterns (DAMPs), including molecules such as DNA and histones, are released into the blood following cell death. DAMPs promote a procoagulant phenotype through enhancement of thrombin generation and platelet activation, thereby contributing to immunothrombosis. Weibel-Palade bodies (WPBs) are dynamic endothelial cell organelles that contain procoagulant and proinflammatory mediators, such as von Willebrand factor (VWF), and are released in response to cell stresses. VWF mediates platelet adhesion and aggregation, and has been implicated as a procoagulant component of the innate immune response. Objective To determine the influence of histones and DNA on WPB release, and characterize their association in models of inflammation. Methods We treated C57BL/6J mice and cultured endothelial cells with histones (unfractionated, lysine-rich or arginine-rich) and DNA, and measured WPB exocytosis. We used inhibitors to determine a mechanism of histone-induced WPB release in vitro. We characterized the release of DAMPs and WPBs in response to acute and chronic inflammation in human and murine models. Results and conclusions Histones, but not DNA, induced the release of VWF (1.46-fold) from WBPs and caused thrombocytopenia (0.74-fold), which impaired arterial thrombus formation in mice. Histones induced WPB release from endothelial cells in a caspase-dependent, calcium-dependent and charge-dependent manner, and promoted platelet capture in a flow chamber model of VWF-platelet string formation. The levels of DAMPs and WPB-released proteins were elevated during inflammation, and were positively correlated in chronic inflammation. These studies showed that DAMPs can regulate the function and level of VWF by inducing its release from endothelial WPBs. This DAMP-WPB axis may propagate immunothrombosis associated with inflammation.

Activation of protein C and thrombin activable fibrinolysis inhibitor on cultured human endothelial cells.

UNLABELLED: ESSENTIALS: It is unknown if thrombin activatable fibrinolysis inhibitor (TAFI) and protein C compete on cells. TAFI and protein C activation on endothelial cells was simultaneously quantified. TAFI and protein C do not compete for activation on endothelial cells. TAFI and protein C are independently recognized by the thrombin-thrombomodulin complex. BACKGROUND: When bound to thrombomodulin (TM), thrombin is a potent activator of protein C (PC) and thrombin activable fibrinolysis inhibitor (TAFI). By binding PC and presenting it to the thrombin-TM complex, endothelial cell PC receptor (EPCR) enhances PC activation. It is unknown whether PC and TAFI compete for the thrombin-TM complex on endothelial cells. OBJECTIVE: To compare PC and TAFI activation on the surface of cultured human endothelial cells in the absence or presence of JRK1535 and/or CTM1009, inhibitory antibodies directed against EPCR and TM, respectively, and to determine whether PC and TAFI compete with each other for activation. METHODS: PC and TAFI activation on endothelial cells were compared, and the effect of PC on TAFI activation and TAFI on PC activation was determined in the absence or presence of JRK1535 and/or CTM1009. RESULTS: In the absence of antibodies, activation of PC was four-fold faster than that of TAFI. Blocking EPCR with JRK1535 resulted in a 53-fold decrease in PC activation and no effect on TAFI activation. Blocking TM with CTM1009 inhibited both TAFI and PC activation. Neither TAFI nor PC competed with each other in the absence or presence of JRK1535. CONCLUSIONS: PC and TAFI are concurrently activated in a TM-dependent manner and do not compete for the thrombin-TM complex, raising the possibility that they interact with distinct activation complexes. EPCR selectively enhances PC activation so that PC and TAFI activation kinetics become comparable on endothelial cells.

Breast cancer chemotherapy induces the release of cell-free DNA, a novel procoagulant stimulus.

BACKGROUND: Thrombosis is a common complication for breast cancer patients receiving chemotherapy. However, the mechanisms by which breast cancer chemotherapeutic agents increase this risk are largely uncharacterized. Nucleic acids released by injured cells may enhance coagulation via the activation of the contact pathway. OBJECTIVES: In this study, we examined the effects of breast cancer chemotherapy agents on the release of cell-free DNA (CFDNA) and its relationship to thrombin generation using in vitro and in vivo methods. METHODS: CFDNA release and thrombin-antithrombin (TAT) levels were measured in plasma of breast cancer patients and healthy mice receiving chemotherapy. Venous whole blood and cultured cells were exposed to chemotherapy and CFDNA release and levels of DNA-histone complexes were measured. The procoagulant activity of isolated CFDNA was measured with calibrated, automated thrombin generation. RESULTS: Breast cancer patients receiving chemotherapy had elevated levels of CFDNA 24 h post-chemotherapy, a time-point at which elevated thrombin-antithrombin levels have been previously reported. Treatment of healthy mice with doxorubicin, epirubicin and 5-fluorouracil increased CFDNA release, with a corresponding elevation in TAT complex formation. Venous whole blood and neutrophils incubated with chemotherapeutic agents had elevated CFDNA in plasma or cell supernatants. In addition, incubation of venous whole blood with chemotherapy decreased histone-DNA complex levels. CFDNA released from epirubicin-treated whole blood significantly elevated thrombin generation in a dose-dependent manner, and involved activation of the contact pathway. CONCLUSIONS: Release of CFDNA from chemotherapy-injured cells may represent a novel mechanism by which thrombosis is triggered in cancer patients.

The chemotherapy metabolite acrolein upregulates thrombin generation and impairs the protein C anticoagulant pathway in animal-based and cell-based models.

BACKGROUND: Thrombosis is a common complication in cancer patients receiving chemotherapy regimens that include cyclophosphamide. However, the mechanisms by which these agents increase this risk are largely uncharacterized. OBJECTIVES: To examine the effects of cyclophosphamide and its metabolite acrolein on procoagulant and anticoagulant pathways in both cell-based and animal-based models. METHODS: Thrombin and activated protein C (APC) generation were measured in defibrinated plasma exposed to acrolein-treated endothelial and smooth muscle cells. Tissue factor (TF) activity was measured on acrolein-treated cells. Cell surface levels of phosphatidylserine, TF, endothelial protein C receptor and thrombomodulin were measured. Healthy BALB/c mice received injections of saline (control), acrolein, or cyclophosphamide; blood was collected, and plasma thrombin-antithrombin (TAT) complex, protein C and APC levels were analyzed. RESULTS: Exposure of acrolein-treated endothelial and smooth muscle cells to defibrinated plasma increased thrombin generation in the plasma. This was associated with enhanced phosphatidylserine exposure and/or increased TF activity on acrolein-treated cells. Despite elevated levels of thrombin generation, plasma APC levels were not elevated. In vivo, treatment of mice with cyclophosphamide and acrolein resulted in elevations of plasma TAT complex levels, whereas APC levels remained low. CONCLUSIONS: This is the first study to examine thrombin generation and the APC pathway in chemotherapy-treated mice. Cyclophosphamide and acrolein appear to upregulate procoagulant pathways, while impairing endogenous anticoagulant pathways. This may explain, in part, the increased risk of thrombosis observed in cancer patients receiving cyclophosphamide-containing chemotherapy.

Chemotherapeutic agents doxorubicin and epirubicin induce a procoagulant phenotype on endothelial cells and blood monocytes.

BACKGROUND: Although chemotherapy is associated with an increased risk of thrombosis, the pathogenic mechanisms by which chemotherapeutic agents exert prothrombotic effects are unclear. OBJECTIVES: In this study we explored the possibility that chemotherapeutic agents doxorubicin, epirubicin, 5-fluorouracil and methotrexate induce a procoagulant phenotype on vascular endothelial cells and/or on blood monocytes. METHODS: Thrombin generation was measured in defibrinated plasma exposed to chemotherapy-treated human umbilical vein endothelial cells (HUVECs). Tissue factor activity assays were performed on chemotherapy-treated HUVECs and blood monocytes. The effects of chemotherapy drugs on phosphatidylserine exposure and the protein C pathway were also measured. RESULTS: Exposure of defibrinated plasma to either doxorubicin- or epirubicin-treated HUVECs resulted in an increase in plasma thrombin generation. The procoagulant activity of doxorubicin- and epirubicin-treated HUVECs reflects an increase in tissue factor activity and phosphatidylserine exposure. Doxorubicin-mediated increase in tissue factor activity is related to increased levels of phosphatidylserine rather than to protein disulfide isomerase activity, and is likely to involve reactive oxygen species generation. Unlike doxorubicin, epirubicin does not have an impact on the protein C anticoagulant pathway. Interestingly, neither methotrextate nor 5-fluorouracil altered endothelial or monocyte hemostatic properties. CONCLUSIONS: These studies suggest that doxorubicin and epirubicin have the greatest 'prothrombotic potential' by virtue of their ability to alter endothelial and monocyte hemostatic properties.